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The use of biophysical methods increases success in obtaining liganded crystal structures.

Chung CW - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale.Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes.Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

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Affiliation: Structural and Biophysical Sciences, GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnelswood Road, Stevenage SG1 2NY, England. cc16943@gsk.com

ABSTRACT
In attempts to determine the crystal structure of small molecule-protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

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Overlay of the crystal structures of the Src SH2 domain with acetylated pYEEI peptide (PDB code 1a1b; in yellow) and the urazole derivative of the YEEI peptide (in green; Chung et al., in preparation). The extensive hydrogen-bonding interactions made by the urazole are shown by dotted green lines. The excellent phosphate mimicry of the urazole heterocycle within this recognition pocket is evident.
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fig5: Overlay of the crystal structures of the Src SH2 domain with acetylated pYEEI peptide (PDB code 1a1b; in yellow) and the urazole derivative of the YEEI peptide (in green; Chung et al., in preparation). The extensive hydrogen-bonding interactions made by the urazole are shown by dotted green lines. The excellent phosphate mimicry of the urazole heterocycle within this recognition pocket is evident.

Mentions: This was indeed the case for our discovery of the urazole moiety as a novel phosphotryosine mimetic. Fragment-based screening using a variety of biophysical methods [e.g. noncovalent mass spectroscopy (Bligh et al., 2003 ▶), fluorescence polarization, scintillation proximity assay and NMR] on the Src SH2 domain identified a number of urazole-containing fragments which competed with phosphotyrosine peptide binding with affinities in the 1–5 mM range. These fragments satisfied many of the criteria required for an ideal pY mimetic, so there was great interest in understanding their binding mode. Their potency was so low that all crystallographic attempts with the isolated fragments proved unsuccessful. However, 1H–15N NMR confirmed that all the urazole moieties bound within the pY pocket. This provided the evidence needed to make a modified recognition peptide with the phosphate group replaced by the urazole heterocycle. This had greater potency (30 µM) and the cocrystal structure of Src SH2 with this urazole peptide was successful. An overlay of the original pYEEI peptide complex with this variant (Fig. 5 ▶) clearly demonstrates the phosphate mimicry of this fragment within the pY pocket (Charifson et al., 1997 ▶; Chung, in preparation).


The use of biophysical methods increases success in obtaining liganded crystal structures.

Chung CW - Acta Crystallogr. D Biol. Crystallogr. (2006)

Overlay of the crystal structures of the Src SH2 domain with acetylated pYEEI peptide (PDB code 1a1b; in yellow) and the urazole derivative of the YEEI peptide (in green; Chung et al., in preparation). The extensive hydrogen-bonding interactions made by the urazole are shown by dotted green lines. The excellent phosphate mimicry of the urazole heterocycle within this recognition pocket is evident.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483471&req=5

fig5: Overlay of the crystal structures of the Src SH2 domain with acetylated pYEEI peptide (PDB code 1a1b; in yellow) and the urazole derivative of the YEEI peptide (in green; Chung et al., in preparation). The extensive hydrogen-bonding interactions made by the urazole are shown by dotted green lines. The excellent phosphate mimicry of the urazole heterocycle within this recognition pocket is evident.
Mentions: This was indeed the case for our discovery of the urazole moiety as a novel phosphotryosine mimetic. Fragment-based screening using a variety of biophysical methods [e.g. noncovalent mass spectroscopy (Bligh et al., 2003 ▶), fluorescence polarization, scintillation proximity assay and NMR] on the Src SH2 domain identified a number of urazole-containing fragments which competed with phosphotyrosine peptide binding with affinities in the 1–5 mM range. These fragments satisfied many of the criteria required for an ideal pY mimetic, so there was great interest in understanding their binding mode. Their potency was so low that all crystallographic attempts with the isolated fragments proved unsuccessful. However, 1H–15N NMR confirmed that all the urazole moieties bound within the pY pocket. This provided the evidence needed to make a modified recognition peptide with the phosphate group replaced by the urazole heterocycle. This had greater potency (30 µM) and the cocrystal structure of Src SH2 with this urazole peptide was successful. An overlay of the original pYEEI peptide complex with this variant (Fig. 5 ▶) clearly demonstrates the phosphate mimicry of this fragment within the pY pocket (Charifson et al., 1997 ▶; Chung, in preparation).

Bottom Line: Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale.Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes.Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural and Biophysical Sciences, GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnelswood Road, Stevenage SG1 2NY, England. cc16943@gsk.com

ABSTRACT
In attempts to determine the crystal structure of small molecule-protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

Show MeSH
Related in: MedlinePlus